Na/K-ATPase (NKA) is the main Na extrusion pathway in cardiac myocytes and therefore is essential in [Na]i regulation. Na and Ca transport are tightly linked in heart via Na/Ca exchange, and the importance of Ca in heart makes understanding [Na]i regulation extremely important. Phospholemman (PLM), a member of FXYD gene family of proteins that associate with and modulate Na/K-ATPase activity. It is also a major target substrate for protein kinase A (PKA) and C (PKC) in heart during sympathetic nervous system activation (and b- and a-adrenergic activation). Here we will test the hypothesis that PLM regulates NKA similar to the way phospholamban regulates SR Ca-ATPase. Aim 1 will examine the roles of phosphorylation of PLM at Ser63 and Ser68 in mediating a- and b-adrenergic effects on NKA activity in intact myocytes (more broadly assessing PKA and PKC effects on PLM-NKA). For these studies we will measure [Na]i and NKA pump current in isolated myocytes from rabbits and mice (including PLM-knockout mice), and complementary measures of PLM phosphorylation. It has been suggested that different NKA isoforms may be localized differently in the sarcolemma and may also interact differently with PLM. Thus, Aim 2 will address the interaction between PLM and NKA and how it is affected by subcellular localization of NKA isoforms and PLM phosphorylation. This will include immunohistochemistry of NKA isoforms, separation of isoforms function by glycoside selectivity, assessment of fluorescence resonance energy transfer (FRET) between CFP-NKA and YFP-PLM and how this is modified by PKA and PKC. Cardiac myocyte Na regulation is perturbed in heart failure and this contributes to altered Ca regulation. In Aim 3 we will address how the adrenergic (or PKA/PKC) dependent modulation of NKA via PLM is altered in heart disease and how it contributes to altered myocyte function. This includes studies in our non-ischemic rabbit heart failure model and complementary work in a rat post myocardial infarction model. The measurements here will focus mainly on isolated myocyte NKA function (modulation by PKA/PKC) and PLM complementary biochemical/molecular measurements. This work will provide comprehensive new information regarding the roles of PLM in cardiac myocytes in normal and failing hearts.